Immunoassay for measurement of reticulocytes, and immunoreactive reagents for use therein

ABSTRACT

The reticulocyte content present in a specimen of red blood cells is quantitatively measured based upon the selective immunoreactivity of the reticulocyte portion of the specimen with a reticulocyte-specific antibody which is immunoreactive with proteinaceous material associated with reticulocytes but not associated with mature red blood cells. Such immunoreactive proteinaceous material may be transferrin, transferrin receptor, transcobalamin II, or transcobalamin II receptor. Various procedures are described for quantitating such selective immunoreactivity, including fluorescent and radioactive detection techniques employing direct or indirect fluorescent or radioactive labeling of the reticulocyte-specific antibody.

BACKGROUND OF THE INVENTION

The invention described herein was made in the course of work under agrant or award from the Department of Health, Education and Welfare.

This invention relates to blood analysis and, more particularly, to thedetermination of the reticulocyte content present in a specimen of redblood cells.

Reticulocytes are newly synthesized red blood cells that have beenreleased from the bone marrow within the preceeding twenty-four toforty-eight hours. In normal circulating blood, the reticulocyte count,i.e., the percentage of reticulocytes based upon the total number of redblood cells, is generally less than two percent. Measurement of thereticulocyte count of a blood specimen is a highly useful diagnostictest, particularly in evaluating the cause of anemia in a patient. Inpatients with anemia, a high reticulocyte count indicates that theanemia is being caused by increased red cell loss, while a lowreticulocyte count indicates the anemia being due to decreased red cellproduction. Also, increased reticulocyte counts found in patients with anormal hemoglobin and hematocrit may alert the physician to increasedred cell loss.

The current methods which are commonly employed for determiningreticulocyte counts utilize special stains which selectively stainnuclear remnants such as RNA which are found in reticulocytes but not inmature red blood cells. Following these time-consuming stainingprocedures, the reticulocytes are manually counted by using a microscopeto determine the percentage of red blood cells that stain asreticulocytes.

The fact that reticulocytes contain RNA indicates that they are able tosynthesize hemoglobin and other proteins. Previously performed bindingstudies have shown that iron bound to transferrin (the major irontransport protein in human plasma) and vitamin B₁₂ bound totranscobalamin II (the major vitamin B₁₂ transport protein in humanplasma) are taken up by reticulocytes but not by mature red blood cells.This suggests that reticulocytes differ from mature red blood cells incontaining receptors for transferrin and transcobalamin II, and, throughsuch receptors, transferrin and transcobalamin II in bound form.

SUMMARY OF THE INVENTION

It is, accordingly, a primary object of the present invention to providean improved method for determining reticulocyte counts which is moreconvenient and less time-consuming to carry out than existing, commonlyemployed procedures.

Another object of the invention is to provide a reticulocyte assay inaccordance with the preceding object, which is readily adaptable for usein conjunction with automated counting techniques.

A further object of the invention is to provide a reticulocyte assay inaccordance with the preceding objects, which is compatible for beingcarried out on-line in conjunction with standard techniques fordetermining total red blood cell count.

Still another object of the invention is to provide novel reagents whichare capable of distinguishing reticulocytes from mature red blood cellswith a high degree of specificity for use in carrying out thereticulocyte assay in accordance with the preceding objects.

The above and other objects are achieved in accordance with the presentinvention by means of an immunoassay procedure which takes advantage ofthe distinctiveness of reticulocytes from mature red blood cells incontaining certain proteinaceous material, such as transferrin,transferrin receptor, transcobalamin II, and transcobalamin II receptor.The immunoassay employs as its primary reagent a reticulocyte-specificantibody which is substantially immunoreactive with the proteinaceousmaterial substantially associated with reticulocytes but notsubstantially associated with mature red blood cells. In employing theimmunoassay for quantitatively measuring the reticulocyte contentpresent in a specimen of red blood cells, an incubation mixture is firstformed comprising the specimen and the reticulocyte-specific antibody.The incubation mixture is then incubated for a period of time sufficientto enable immunoreaction to occur. This results in thereticulocyte-specific antibody forming an immunoreaction productselectively with the reticulocyte portion of the specimen via theimmunoreactive proteinaceous material. Thereafter, the reticulocyteportion of the specimen is quantitatively measured based upon the extentof the immunoreaction, which may be determined in any of severaldifferent ways. Particularly suitable as quantitating techniques arefluorescent or radioactive detection techniques employing direct orindirect fluorescent or radioactive labeling of thereticulocyte-specific antibody.

The immunoassay of the present invention is readily adaptable for use inconjunction with standard manual or automated fluorescent or radioactivecounting systems. Furthermore, the immunoassay together with itsassociated counting system is readily compatible with being carried outon-line in conjunction with standard techniques for determining totalred blood cell count, thereby enabling an integrated system fordetermining both the total red blood cell count and the reticulocytecount of a blood specimen.

DESCRIPTION OF PREFERRED EMBODIMENTS

The reticulocyte-specific antibody employed as the primary reagent inthe immunoassay of the present invention is defined as one which issubstantially immunoreactive with proteinaceous material substantiallyassociated with reticulocytes but not substantially associated withmature red blood cells. Such proteinaceous material is most suitablyselected from the group consisting of transferrin, transferrin receptor,transcobalamin II, transcobalamin II receptor, and combinations thereof.

The antibody reagent may suitably be prepared by standardantibody-producing techniques in any suitable animal, most typically arabbit, starting with a purified form of the proteinaceous material withwhich the antibody is to be immunoreactive. Purified human transferrinsuitable for use in antibody production is commercially available, forexample, from Sigma Chemical Company, St. Louis, Missouri. Purifiedhuman transcobalamin II and purified human transcobalamin II receptorsuitable for use in antibody production may suitably be prepared by theprocedures described by Allen, et al., J. Biol. Chem., Vol. 247, pp.7709-7717 (1972) (Human transcobalamin II); and Seligman, et al., J.Biol. Chem., Vol. 253, pp. 1766-1772 (1978) (Human transcobalamin IIreceptor), both of which are incorporated herein by reference.

A procedure for the preparation of purified human transferrin receptor,isolated from human placenta, and suitable for use in antibodyproduction, has been developed by the present inventors. Suchpurification procedure, and the characterization of the purified humantransferrin receptor obtained thereby, are described in detail in apublished paper by Seligman, et al., appearing in J. Biol. Chem., Vol.254, No. 20, pp. 9943-9946 (October 25, 1979), which paper isincorporated herein by reference. In carrying out such purificationprocedure, a crude solubilized transferrin receptor preparation is firstobtained by mixing a human placental homogenate in physiologic bufferwith approximately one percent Triton X-100, followed by centrifugationand recovery of the crude solubilized transferrin receptor preparationas the supernatant. This crude preparation also contains transferrin,both in free form and bound to the transferrin receptor. The transferrinreceptor-transferrin complex is then precipitated from the crudepreparation by mixing with ammonium sulfate, leaving the freetransferrin remaining in the supernatant. The transferrinreceptor-transferrin complex is then extracted from the ammonium sulfateprecipitate with 0.1 percent Triton X-100 in physiologic buffer, andsubjected to immunochromatography on an antihuman transferrin-Sepharosecolumn, which binds the transferrin receptor-transferrin complex. Thetransferrin receptor substantially free of transferrin is then elutedfrom the immunochromatography column with en elution buffer composed of20 mM glycine/NaOH, pH 10.0, 500 mM NaCl, and 0.5 percent Triton X-100.The transferrin receptor eluted from the immunochromatography column isthen subjected to affinity chromatography on a humantransferrin-Sepharose column, from which it is eluted with an elutionbuffer composed of 50 mM glycine/NaOH, pH 10.0, 1 M NaCl, and 1.0percent Triton X-100. The transferrin receptor eluted from the affinitychromatography column is then concentrated by ammonium sulfateprecipitation and extraction from the ammonium sulfate precipitate with0.1 percent Triton X-100 in physiologic buffer, to form the finalpurified transferrin receptor preparation. This purification procedureresults in the isolation of human transferrin receptor having a purityof greater than 95 percent from a human placental starting materialcontaining the transferrin receptor in a concentration of less than 0.01percent.

Preparation of the antibody reagent from the purified proteinaceousmaterial may be carried out by standard techniques well known in theart, i.e., by injection of any suitable animal, such as rabbits, goats,sheep, etc., with the purified proteinaceous material, and subsequentcollection of the resulting antiserum. In a typical procedure, aninjectable preparation of the purified proteinaceous material isprepared in physiologic buffer mixed with an equal volume of Freund'scomplete adjuvant. Rabbits are injected with this preparation, eachrabbit receiving 100 μg of the proteinaceous material on Day 1, given asinjections of 25 μg into each foot pad. The rabbits are boosted on Day30 by injections given in the same manner, except that the amount ofproteinaceous material injected per rabbit is reduced to 40 μg. Therabbits are bled on Day 40. The blood is allowed to clot at roomtemperature for one hour, and then stored at 4° C. for sixteen hours.The antisera are then collected by centrifugation, e.g., at 20,000×g forthirty minutes, and stored at -20° C. Monoclonal antibodies may alsoprepared using a technique such as mouse hybridomas.

For use as the antibody reagent in carrying out the immunoassay of thepresent invention, the antisera obtained in the above manner maysuitably be employed as such. Alternatively, purified antibody can beobtained from the antisera by conventional means, for example, ammoniumsulfate precipitation or affinity chromatography on antigen-Sepharose,and the antibody in thus-purified form may be employed as the antibodyreagent.

The form of the specimen of red blood cells whose reticulocyte count isbeing measured depends upon the particular reticulocyte-specificantibody reagent being employed. Blood serum normally contains asignificant amount of free, unbound transferrin and transcobalamin II.Due to this fact, when the antibody reagent is anti-transferrin antibodyor anti-transcobalamin II antibody, the red blood cells must beseparated from the blood serum, washed with physiologic buffer (e.g.,phosphate-buffered saline or 10 mM potassium phosphate, pH 7.5, 150 mMNaCl), and then resuspended in physiologic buffer. On the other hand,when the antibody reagent is anti-transferrin receptor antibody oranti-transcobalamin II receptor antibody, the red blood cells need notbe washed and can be subjected to the immunoassay in blood serumsuspension form. For this reason, coupled with its higher sensitivitywith respect to reticulocytes in comparison with anti-transcobalamin IIreceptor antibody, the preferred antibody reagent for use in theimmunoassay of the present invention is anti-transferrin receptorantibody.

In carrying out the immunoassay in accordance with the presentinvention, an incubation mixture is first formed comprising the specimenof red blood cells whose reticulocyte content is to be measured, and thereticulocyte-specific antibody reagent. The relative proportions ofthese components of the incubation mixture may vary over a rather broadrange. In general, with an incubation mixture containing approximately2×10⁸ red blood cells, the antibody reagent should be employed in anamount providing, broadly, from about 0.0001 to about 100, andpreferably, from about 0.1 to about 10 μg of the reticulocyte-specificantibody. As a general rule, the reticulocyte-specific antibodyconcentration in the antisera as collected from the animal will beapproximately 1 μg/μl. Thus, when the antisera is used directly as theantibody reagent without further antibody purification, from about0.0001 to about 10 μl of the antisera will provide the requisite amountof reticulocyte-specific antibody. If desired, the antisera can bediluted with physiologic buffer to a volume which is most convenient foruse in forming the incubation mixture. For example, a suitableincubation mixture may conveniently be formed by adding to 50 μl of redblood cell suspension the requisite amount of reticulocyte-specificantibody in a volume of 10 to 100 μl.

The thus-formed incubation mixture is then incubated for a period oftime sufficient to enable immunoreaction to occur. The requisite timeperiod will vary, depending upon the temperature at which the incubationis carried out, with lower incubation temperatures requiring longerincubation periods. In general, the incubation period may vary fromabout five minutes to about twelve hours at incubation temperatureswithin the range of from about 4° to about 37° C. Particularly suitableincubation schedules are thirty minutes at 37° C. or two hours at roomtemperature.

The immunoassay of the present invention is based upon the selectiveimmunoreactivity of the reticulocyte portion of the red blood cellspecimen with the reticulocyte-specific antibody reagent via theimmunoreactive proteinaceous material which is substantially associatedwith the reticulocytes but not substantially associated with the maturered blood cells. Quantitating such selective immunoreactivity based uponthe extent of the immunoreaction serves as a quantitative measurement ofthe reticulocyte portion of the specimen. Any one of several differentquantitating techniques may be employed in conjunction with theimmunoassay for making such quantitative measurement.

The quantitating technique employed with the immunoassay of the presentinvention may be based upon the amount of the immunoreaction productformed between the reticulocyte portion of the red blood cell specimenand the reticulocyte-specific antibody reagent. This may be determinedas the amount of bound antibody present in the immunoreaction product,which may be quantitated by standard manual or automated fluorescent orradioactive detection techniques well known in the art employing director indirect fluorescent or radioactive labeling of thereticulocyte-specific antibody. Thus, the reticulocyte-specific antibodywhen incorporated into the incubation mixture may be influorescent-labeled form, labeled with a suitable fluorescent labelingagent such as, for example, fluorescein or rhodamine, or it may be inradioactive-labeled form, labeled with a suitable radioactive labelingagent such as, for example, ¹²⁵ I or ¹³¹ I.

Alternatively, the reticulocyte-specific antibody when incorporated intothe incubation mixture may be in unlabeled form, and labeling may beeffected subsequent to the immunoreaction by incubating theimmunoreaction product with a fluorescent-labeled or radioactive-labeledsubstance which is bindable to the immunoreacted reticulocyte-specificantibody. Such antibody-bindable substance will typically be a secondantibody directed against the reticulocyte-specific antibody, i.e.,raised to the globulins of the animal source of thereticulocyte-specific antibody (anti-IgG). Alternatively, theantibody-bindable substance may be, for example, Staphylococcal Aprotein, a protein which is known to bind to antibodies.

With either the direct or indirect fluorescent or radioactive labelingapproach described above, the fluorescence or radioactivity, as the casemay be, exhibited by the immunoreaction product may be quantitated bystandard manual or automated fluorescent or radioactive countingtechniques well known in the art, thereby providing a quantitativemeasurement of the reticulocyte content of the red blood cell specimen.

Another technique for determining the amount of the immunoreactionproduct formed between the reticulocyte-specific antibody and thereticulocyte portion of the red blood cell suspension, involves the useof complement to the reticulocyte-specific antibody. Such complement maybe added to the incubation mixture, and the amount of the immunoreactionproduct formed may then be determined by measurement of the resultingcomplement-mediated effects. For example, this procedure might result inswelling of the reticulocytes, thus allowing for differentiation frommature red blood cells by size. Also, this procedure might causecomplete complement-mediated hemolysis of the reticulocytes, which couldbe quantitated in an automated system by measuring the amount ofhemoglobin released or by the loss of a subpopulation of larger sizedcells noted before the cells are treated.

The quantitative measurement of the reticulocyte portion of the specimenmay also be based upon the amount of the immunoreactive proteinaceousmaterial present in the specimen. For carrying out the immunoassay ofthe present invention in this manner, the incubation mixture will alsocontain a known amount of the immunoreactive proteinaceous material inaddition to that present in the specimen and in radioactive-labeledform, and the amount of the immunoreactive proteinaceous materialpresent in the specimen will be determined by standard radioimmunoassaytechniques well known in the art.

The immunoassay of the present invention, particularly when employed inconjunction with the fluorescent or radioactive detection techniquesdescribed above, can be readily adapted to an automated system forreticulocyte counting by adding the appropriate reticulocyte-specificantibody to a blood cell specimen subjected to an existing flow systemused for cell counting. All red blood cells would be counted in the flowsystem, and a fluorescent or radioactive detector could be situatedon-line down stream from the cell counter. Then, using existingcomputerized techniques, the number of fluorescent or radioactive cellscould be tabulated, and the percentage of reticulocytes quantitated.

The invention is further illustrated by way of the following examples.

EXAMPLE 1

This example illustrates the preparation of purified human transferrinreceptor, isolated from human placenta.

A placental homogenate was prepared by cutting two placentas weighing atotal of 1500 g into approximately 40 g pieces, and homogenizing them ina Waring Blendor for 30 seconds in 1.5 volumes of 10 mM KPO₄, pH 7.5,containing 150 mM NaCl. The mixture was centrifuged, and the pelletswere resuspended in 1500 ml of the same buffer and frozen. The frozenhomogenate could be stored for up to 3 days with no loss of receptoractivity.

The resuspended pellet was thawed and 500 ml of 10 mM KPO₄, pH 7.5,containing 150 mM NaCl and 4 percent Triton X-100 was added.Solubilization was then continued utilizing homogenization with a WaringBlendor, sonication, and centrifugation. Crude solubilized transferrinreceptor preparation was recovered as the supernatant.

Solid ammonium sulfate, 31.5 g/100 ml, was added to the supernatant fromthe previous step. After stirring for 10 minutes at 4° C., the samplewas centrifuged at 20,000×g for 20 minutes at 4° C. Since the TritonX-100 present caused flotation of the precipitate, clear centrifugebottles were used, and the supernatant was aspirated with a vacuumsuction device. The precipitate was resuspended in 600 ml of 10 mM KPO₄,pH 7.5, containing 150 mM NaCl and 0.1 percent Triton X-100 and dialyzedagainst 4 liters of the same solution for 16 hours. The dialysate waschanged at 4 and 12 hours.

The dialyzed ammonium sulfate preparation from the previous step wascentrifuged in 200-ml portions at 16,000×g for 20 minutes. Thesupernatant was then applied to a column (2×30 cm) containing 60 ml ofantihuman transferrin-Sepharose equilibrated in 10 mM KPO₄, pH 7.5, 150mM NaCl (equilibration buffer), and 0.1% Triton X-100 at 4° C. It wasnecessary to centrifuge the dialyzed ammonium sulfate preparation justbefore applying it to the column to prevent the slow formation ofprecipitates which would not allow the flow rate of 50 ml/h to bemaintained. After 200 ml of solution had been applied, the column waswashed with a variety of solutions in the following order: wash (1) 200ml of equilibration buffer; wash (2) 100 ml of 10 mM KPO₄, pH 7.5, 500mM NaCl; wash (3) 100 ml of equilibration buffer; wash (4) 200 ml of 20mM glycine/NaOH, pH 10.0, 500 mM NaCl, and 0.5% Triton X-100; and wash(5) 100 ml of 10 mM glycine/HCl, pH 2, 150 mM NaCl. The flow rate forwashes 1 to 3 was 100 ml/h and 50 ml/h for washes 4 and 5. Wash 4contained the bulk of the receptor activity. Wash 5 was performed toremove all the bound transferrin so the affinity column could be reused.Solid ammonium sulfate (31.5 g/100 ml) was added to wash 4, and thesolution was stirred and centrifuged, the supernatant was aspirated, andthe pellet was resuspended in 100 ml of equilibration buffer anddialyzed as described above in the previous ammonium sulfateprecipitation step.

The dialyzed ammonium sulfate pellet from the previous step was appliedto a column (1.5×15 cm) containing 15 ml of human transferrin-Sepharosein equilibration buffer. The column was then washed with the followingsolutions: wash (1) 100 ml of equilibration buffer; wash (2) 40 ml of 10mM KPO₄, pH 7.5, 1 M NaCl; wash (3) 20 ml of equilibration buffer; andwash (4) 40 ml of 50 mM glycine/NaOH, pH 10.0, 1 M NaCl, and 1.0% TritonX-100. Wash 4, which contained the bulk of receptor activity, wasconcentrated to 4 ml using ammonium sulfate precipitation, and dialyzedagainst equilibration buffer as described above in the previous ammoniumsulfate precipitation steps, to obtain the final transferrin receptorpreparation.

The final transferrin receptor preparation contained 1.2 mg of protein.Assays for immunoreactive transferrin indicated that the preparationcontained only 15 μg of transferrin which could be removed by passingthe solution over an antihuman transferrin-Sepharose column.

The final transferrin receptor preparation gave a single band onpolyacrylamide disc gel electrophoresis which coincided with a singlepeak of ¹²⁵ I-transferrin binding ability, and stained for both proteinand carbohydrate. On sodium dodecyl sulfate-polyacrylamide gelelectrophoresis, the transferrin receptor had a molecular weight of150,000, and dissociated into 90,000, 60,000 and 30,000 molecular weightcomponents after reduction with 2-mercaptoethanol.

EXAMPLE 2

This example illustrates the preparation in rabbits of anti-humantransferrin receptor antibody from the purified human transferrinreceptor prepared in Example 1.

The purified human transferrin receptor preparation as prepared inExample 1, in physiologic buffer, was mixed with an equal volume ofFreund's complete adjuvant. Each rabbit received 1 ml of this mixture,containing 100 μg of transferrin receptor, on Day 1, given as injectionsof 0.25 ml into each footpad. Rabbits were boosted on Day 30 byinjections given in the same manner, except that the amount oftransferrin receptor injected per rabbit was reduced to 40 μg. Rabbitswere bled on Day 40. The blood was allowed to clot at room temperaturefor 1 hour, and then was stored at 4° C. for 16 hours. The antisera werethen collected by centrifugation at 20,000×g for 30 minutes and storedat -20° C.

The thus prepared anti-human transferrin receptor antibody was found toinhibit the binding of ¹²⁵ I-transferrin to purified human transferrinreceptor. The antibody gave a single precipitin line against thepurified transferrin receptor which cross-reacted with a pattern ofidentity with solubilized placental homogenate and solubilized red cellmembranes from a patient with a 20 percent reticulocyte count; noprecipitin line was seen with solubilized red cells from a subject withless than 2 percent reticulocytes. This information indicates that thetransferrin binding protein that was isolated is the placental cellsurface transferrin receptor and shows that the placental transferrinreceptor is immunologically related to the transferrin receptor on thereticulocyte membrane. The data also suggests that immunoreactivereticulocyte transferrin receptor is altered or lost from the cellsurface as the reticulocyte matures into a normal red cell.

EXAMPLE 3

This example illustrates the reticulocyte immunoassay of the presentinvention, carried out as a double antibody immunofluorescent technique.

A specimen of red blood cells (2×10⁸) known to have a 20 percentreticulocyte count was washed in physiologic buffer (10 mM KPO₄, pH 7.5,150 mM NaCl), then suspended in 50 μl of the physiologic buffer, andthen incubated with 10 μl of the rabbit anti-human transferrin receptorantisera prepared as in Example 2, above. The incubation was carried outfor 30 minutes at 37° C. The cells were again washed in physiologicbuffer, resuspended in 50 μl of the physiologic buffer, and reincubatedwith 100 μl of the second antibody, which was fluorescein-labelled goatanti-rabbit globulin, for 30 minutes at 37° C. After a final wash inphysiologic buffer, the cells were examined under microscopy using botha phase and fluorescent light source. The phase field showed a clump ofreticulocytes surrounded by mature red blood cells. The same field usingthe fluorescent source showed that only the reticulocytes exhibitedmembrane fluorescence, whereas the mature red blood cells did not. Thefluorescence was not quenched when endogeneous transferrin was strippedfrom the reticulocytes, or when the antisera was previously absorbedwith excess transferrin.

The above procedure was repeated, but this time employing as the firstantibody rabbit anti-human transferrin antisera. Again, the comparisonbetween the phase source and the fluorescent source showed that thereticulocytes exhibited membrane fluorescence, whereas the mature redblood cells did not. This time, however, the fluorescence was quenchedwhen endogenous transferrin was stripped from the reticulocytes, or whenthe antisera was previously absorbed with excess transferrin.

EXAMPLE 4

The double antibody immunofluorescent technique as described in Example3, above, was used for determining the reticulocyte counts of bloodspecimens obtained from a patient having pernicious anemia diagnosed bylow serum B₁₂ and an abnormal Schilling test. With patients of thistype, an injection of vitamin B₁₂ results in a daily increase inreticulocyte count, with peak reticulocyte response expected 4 to 5 daysfollowing the injection. The patient was given his first injection ofvitamin B₁₂ on Day 1. Reticulocyte counts were measured on the daypreceding the injection (Day 0) and daily for the 4 days subsequent tothe injection (Days 2 to 5). Reticulocyte counts employing the doubleantibody immunofluorescent technique of the present invention weredetermined as the percent fluorescent cells based upon the counting of1,000 red blood cells. The reticulocyte counts were the same employingeither anti-human transferrin receptor antisera or anti-humantransferrin antisera as the first antibody in the double antibodyimmunofluorescent technique. For purposes of comparison, simultaneousreticulocyte counts were also performed using standard supravitalstaining techniques and also counting 1,000 red blood cells. Theresulting reticulocyte counts obtained are set forth in Table I, below,which also lists for comparison purposes reticulocyte counts performedon a normal subject.

                  TABLE I                                                         ______________________________________                                                 Reticulocyte Count                                                              Immunofluorescent                                                                             Supravital Stain                                   Day        Technique       Technique                                          ______________________________________                                        0          0.5             1.7                                                2          2.0             5.0                                                3          6.0             9.0                                                4          15.0            19.0                                               5          16.0            22.0                                               Normal     0.2             0.8                                                ______________________________________                                    

It can be seen from Table I, that reticulocyte counts employing theimmunofluorescent technique in accordance with the present invention aresomewhat lower than those obtained using the standard supravitalstaining technique, although the results are still comparable. It islikely that with a more sensitive fluorescent detector, reticulocytecounts using the immunofluorescent technique would be the same as withthe standard supravital staining method.

When the double antibody immunofluorescent technique described above wascarried out employing as the first antibody either anti-humantranscobalamin II antibody or anti-human transcobalamin II receptorantibody, the results were comparable, but with much lower levels offluorescence being observed on the reticulocytes. Again, it is likelythat an improved level of fluorescence would be obtained utilizing amore sensitive fluorescent detector.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A method for thequantitative assay of the reticulocyte content present in a specimen ofred blood cells, comprising the steps of:(a) forming an incubationmixture comprising said specimen and a reticulocyte-specific antibodywhich is immunoreactive with proteinaceous material substantiallyassociated with reticulocytes but not substantially associated withmature red blood cells; (b) incubating said incubation mixture for aperiod of time sufficient to enable immunoreaction to occur, wherebysaid reticulocyte-specific antibody forms an immunoreaction productselectively with the reticulocyte portion of said specimen via saidimmunoreactive proteinaceous material; and (c) quantitatively measuringthe reticulocyte portion of said specimen based upon the extent of saidimmunoreaction.
 2. The method of claim 1, wherein said immunoreactiveproteinaceous material is selected from the group consisting oftransferrin, transferrin receptor, transcobalamin II, transcobalamin IIreceptor, and combinations thereof.
 3. The method of claim 2, whereinsaid immunoreactive proteinaceous material is transferrin receptor. 4.The method of claim 1, wherein the quantitative measurement of thereticulocyte portion of said spcimen is based upon the amount of saidimmunoreaction product formed.
 5. The method of claim 4, wherein saidreticulocyte-specific antibody is in fluorescent-labeled form, and theamount of said immunoreaction product formed is determined byfluorescent detection techniques.
 6. The method of claim 4, wherein saidreticulocyte-specific antibody is in radioactive-labeled form, and theamount of said immunoreaction product formed is determined byradioactive detection techniques.
 7. The method of claim 4, includingthe further step subsequent to step (b) of incubating saidimmunoreaction product with a fluorescent-labeled substance which isbindable to the immunoreacted reticulocyte-specific antibody, and theamount of said immunoreaction product formed is determined byfluorescent detection techniques.
 8. The method of claim 7, wherein saidfluorescent-labeled substance is a fluorescent-labeled second antibodydirected against said reticulocyte-specific antibody.
 9. The method ofclaim 4, including the further step subsequent to step (b) of incubatingsaid immunoreaction product with a radioactive-labeled substance whichis bindable to the immunoreacted reticulocyte-specific antibody, and theamount of said immunoreaction product formed is determined byradioactive detection techniques.
 10. The method of claim 9, whereinsaid radioactive-labeled substance is a radioactive-labeled secondantibody directed against said reticulocyte-specific antibody.
 11. Themethod of claim 4, wherein complement to said reticulocyte-specificantibody is added to said incubation mixture, and the amount of saidimmunoreaction product formed is determined by measurement of theresulting complement-mediated effects.
 12. The method of claim 1,wherein the quantitative measurement of the reticulocyte portion of saidspecimen is based upon the amount of said immunoreactive proteinaceousmaterial present in said specimen.
 13. The method of claim 12, whereinsaid incubation mixture contains a known amount of said immunoreactiveproteinaceous material in addition to that present in said specimen,said known amount being in radioactive-labeled form, and the amount ofsaid immunoreactive proteinaceous material present in said specimen isdetermined by radioimmunoassay techniques.